A pilot study of the ageing urinary microbiome Eur Urol Suppl 2013;12;e161
Williams J.P.1, Lewis D.1, Brown R.2, Marchesi J.R.2, Drake M.J.1 1
Bristol Urological Institute, Dept. of Urology, Bristol, United Kingdom, 2Cardiff University, School of Biosciences,
Bristol, United Kingdom INTRODUCTION & OBJECTIVES: Humans are colonised by large collections of microbes known the microbiome. The urinary bladder has traditionally been considered sterile. However it has recently become evident that urine from healthy individuals could contain extensive numbers of bacteria not detected by routine clinical microbiology laboratory techniques. The 16s rRNA gene is a commonly used genetic marker for the presence of bacterial DNA. The use of 16s rRNA gene sequencing can detect the presence of bacteria which would not be identified through routine culture methods. The aim of this pilot study was to identify the total urinary microbiota using non culture methods in a number of healthy individuals of differing ages. MATERIAL & METHODS: Healthy individuals provided a mid-stream urine sample by the clean-catch method. DNA was extracted by bead beating in a lysis buffer containing detergent followed by alcohol precipitation. The 16sRNA gene, a marker for the presence of bacteria was the target for PCR amplification with resulting amplicons analysed by the 454 pyrosequencing high-density system. Sample bacterial load was quantified by 16S rRNA DNA qPCR of the urine DNA extracts. Sample DNA was calibrated against standard DNA obtained from an enumerated Escherichia coli liquid culture extracted by the urine DNA protocol. Urine bacterial load was expressed as colony forming units /ml (cfu/ml) equivalent. Raw sequencing reads were quality trimmed using the RDP Pyrosequencing Pipeline. Pyrosequencing reads were aligned with Infernal and associated covariance models obtained from the Ribosomal Database Project Group. Mothur was used for taxonomic grouping . RESULTS: Urine samples from 16 asymptomatic individuals were analysed (10 female and 6 male). Age range of the group was 26-90 years. Mean age was 61.1 years. For females aged 20 -49 (n= 3) the number of different genera present was high (n=48) as was the predicted number of cfu/ ml (>103) for each genus. The number of different genera was 36 for the group aged 50-69 (n=3) and 43 for women aged 70+ (n=4). Notably, however, at the age of 70+ the predicted numbers of bacteria present was much less than at ages 20-49. For males, aged 20-49 (n=2) the number of different genera (n=4) was considerably less than the equivalent female group but once again the predicted number of bacteria of each genus was high. For older men, the number of different genera increased from 9 (ages 50-69; n=2) to 47 (ages 70+; n=2). The predicted number of bacteria present for each genus at age 70+ in comparison to ages 20-49 was much less. CONCLUSIONS: The healthy bladder microbiota changes with ageing, both in complexity and in the abundance of
bacteria present. In both men and women, there were large numbers of bacterial genera, but small numbers of bacteria predicted. Many of these bacteria would be missed by standard culture methods. Understanding how the aging process shapes the healthy bladder microbiota is necessary for future studies in predicting the effect of changes in the bladder microbiota on the development of disease.